The invention relates to methods and devices for performing specific binding assays, in particular, for detecting the presence of an analyte in a fluid sample.
Detection of a particular analyte (e.g., an antigen, such as a pathogen or a hormone or a single-stranded nucleic acid target) in a fluid sample may be accomplished using a variety of binding assays, e.g., immunoassays or DNA hybridization assays. Generally, such an assay involves reaction of the test sample with a specific binding reagent (e.g., a specific antibody) and with a reagent which facilitates the direct or indirect quantitative measurement of the amount of the analyte of interest in the test sample. In one particular example known as an Enzyme-linked Immunosorbent Assay or ELISA, an antibody covalently bound to an enzyme (e.g., horseradish peroxidase) is reacted with a test sample and the presence of an analyte is assayed by reaction of the immunocomplex with substrate (e.g., 4-chloro-1-naphthol) followed by measurement of the colored end product.
Coleman (U.S. Pat. No. 3,799,742; 1974) reports an immunoassay which involves the breaking of two membranes to allow a fluid sample to flow into a chamber containing specific binding reagents (e.g., an antibody specific for the analyte). The presence of the analyte is monitored by a color reaction.
Bauer et al. (U.S. Pat. No. 3,811,840; 1974) report the use of specific binding reagents immobilized on an absorbent wick. The wick is dipped into an analyte-containing sample, facilitating the migration of the sample into the wick; the presence of analyte is detected by a color reaction.
Neremberg (U.S. Pat. No. 3,645,687; 1972) reports an immunoassay similar to that of Bauer et al., except that the analyte-containing sample is applied with a capillary tube.
Dafforn et al. (Clin. Chem. 36: 1312, 1990) report an immunoassay for HIV antibody detection. Latex microspheres coated with antigen are embedded in the fibers of a wick; a human serum sample, applied to the wick, migrates downstream (by capillary action) contacting the antigen. Crushing of a substrate ampule releases substrate into a sponge (located upstream of the sample entry port). When saturated, the sponge expands to contact the wick, and substrate is slowly transported into the wick. Simultaneous to substrate release, a protein-A-enzyme conjugate solution is manually added to the wick downstream of the sample entry port. Slow release of the substrate by the sponge allows time for conjugate reaction with the immobilized antibody-antigen complex. Complexes are detected by color reaction.
Li et al. (Analytical Biochem. 166: 276, 1987) report a one-step immunoassay in which a paper support containing immobilized antibodies and enzyme is dipped into a sample containing substrate, a color indicator, and an enzyme inhibitor, as well as the analyte to be detected. Capillary action draws the sample up the paper, the inhibitor (present in a finite quantity) migrates out of the reactive zone, and color development is assayed. The height of the color bar is proportional to the analyte concentration.
Zuk et al. (U.S. Pat. No. 4,435,504; 1984 and Clin. Chem. 31: 1144, 1985) report a competitive immunoassay using a dry paper strip containing immobilized antibody. The strip is dipped first into a solution containing both the analyte and an enzyme-analyte conjugate and then completely immersed into a solution containing substrate and a color developer. The height of the color bar is proportional to the analyte concentration.
Deutsch et al. (U.S. Pat. No. 4,094,647; 1980) report a competitive immunoassay in which a sample analyte is blotted onto an absorbent test strip. The strip is dipped into a developing fluid which transports the analyte along the test strip (by capillary action), facilitating contact first with labelled analyte and then with immobilized analyte-specific antibody. Unbound reagents are transported farther along the test strip. Labelled analyte and sample analyte compete for binding to the immobilized antibody; the amount of label measured at the site of immobilized antibody is inversely proportional to the quantity of analyte in the sample.
Cole et al. (U.S. Pat. No. 3,246,339; 1981) report the use of an absorbent reservoir to draw off excess liquid from the test wells of an assay device; analyte is trapped on the test wells' porous membrane surface and detected.
Bagshawe (U.S. Pat. No. 3,888,629; 1975) report the use of an absorbent reservoir to promote filtration of a fluid sample through a matrix pad containing dried specific binding reagent.
Tom et al. ((U.S. Pat. No. 4,366,241) reports an immunoassay device having two bibulous zones, an analyte binding partner being non-diffusively fixed in a first zone (the "immunoabsorbing zone"), and the second zone being a reservoir zone which is either directly or indirectly in liquid-receiving relationship with the first zone to pull liquid through and out of the first zone.
Brooks et al. (WO90/05906) reports a test device which includes (i) a reaction zone capable of retaining a detectable assay product, (ii) a control absorbent in liquid-transferring contact with the reaction zone which meters a predetermined flow of sample or binding reagents through the reaction zone, and (iii) an absorbent reservoir which speeds flow of fluid through the control absorbent.